Clostridium difficile infections (CDI) have become more difficult to treat due to the rise of hypervirulent strains that have increased morbidity as well as mortality and the likelihood of persistence and relapse in infected patients. To address the dire need for new anti-difficile agents, we explored the premise that nature utilizes good bacteria (i.e. probiotics), including Lactobacillus spp., to suppress gut pathogens such as C. difficile by producing novel antimicrobials, which were optimized by evolution to the microenvironment of the gut. In this regard, we explored the potential for developing reutericyclin from Lactobacillus reuteri as a natural anti- difficile agent. Probiotics are emerging as alternate treatments for CDI. However, there is ongoing debate on whether live probiotics may be used treat severe CDI, especially in immunocompromised patients. We therefore hypothesize that developing the antimicrobial produced by the probiotic specie would harness one of its natural therapeutic properties for localized killing of C. difficile in the gut, providing a more reliable and efficacious treatment strategy. Application of this concept to our studies on reutericyclin revealed that it has impressive antimicrobial and pharmacological properties for treating CDI. These include: rapid killing of nongrowing, toxin- producing stationary phase C. difficile;a novel mechanism of action specific to the bacterial membrane;a narrow spectrum of activity;lack of cytotoxicity against gut epithelia;stability to proteolysis;ability to achieve high non- absorbed concentrations in gut for killing;a low molecular weight and ease of synthesis that will allow advanced chemical optimization. Importantly, the killing of toxin-producing stationary phase cells is not shown by currently prescribed antibiotics vancomycin and metronidazole, which only kill actively growing C. difficile. We believe that this proposal for developing probiotic-derived reutericylcin derivatives to treat CDI is highly innovative and will be achieved through three iterative aims: (i) Synthesis of an expanded sets of reutericyclin analogs to optimize anti-difficile activity and increase affinity for the membrane target site;(ii) Lead development and characterization involving the stepwise progression of compounds through three stages of tests that include antimicrobial assessment, pharmacokinetic testing, toxicologic and in vivo efficacy experiments. Compounds meeting the selection criteria of the tests will move onto the next stage such that a lead candidate is obtained with potent in vivo efficacy and excellent safety profile;(iii) Mode of action studies to explore the fundamental antibacterial effects of targeting the clostridial membrane. The long-term goal of this project is to develop optimized lead analogs of probiotic derived reutericyclin that exhibit novel modes of action at the membrane target and have characterized antibiotic properties that would allow their progression into advanced preclinical studies as candidates for treating CDI. PUBLIC HEALTH RELEVANCE: There is a need for novel treatments for Clostridium difficile infections, which are now a major cause of morbidity and mortality among elderly patients in hospital settings in the United States. To address this need, we propose to optimize and develop derivatives of the probiotic-derived reutericyclin that is obtained from Lactobacillus reuteri. Because these agents rapidly kill C. difficile they may prevent the relapse of infection.